We propose the first experimental studies of the historical origin and elaboration of molecular complexes. Virtually all proteins assemble with specific molecular partners into precise geometric arrangements, but we know little about the genetic and structural mechanisms by which these complexes evolved or the evolutionary forces that explain their origin, elaboration, and long-term persistence. We will combine ancestral protein reconstruction with biochemical, structural, and functional experiments to reconstruct the evolution of molecular complexes in three model protein families, enabling us to formulate and test general hypotheses about the evolutionary causes and consequences of changes in stoichiometry, allostery, and specificity. All three protein families are biologically essential, experimentally tractable, and exemplify distinct questions. The models are: 1) hemoglobin, the major oxygen carrier in vertebrates, a heterotetramer that is biochemistry's iconic case of an allosterically regulated molecular complex; 2) citrate synthase, an essential metabolic enzyme that is a dimer in some lineages and an allosterically regulated hexamer in others, which provides a rich case-study of the evolutionary relationship and long-term persistence of allostery and complexity; and 3) steroid hormone receptors, a family of transcription factors that regulate vertebrate reproduction and development and which evolved after gene duplication to specifically assemble as homodimers, each with distinct regulatory functions. The project will address these questions: 1) How and why do complexes originate and increase in stoichiometry from simpler forms? 2) What genetic and biophysical mechanisms mediate evolution of new and specific interfaces? 3) By what mechanisms did allostery evolve? 4) Does selection for oligomer-associated functions account entirely for the emergence and long-term persistence of molecular complexes? Or did substitutions compatible only with the assembled form occur neutrally and entrench the complex, creating an evolutionary ratchet towards greater complexity? And 5) After duplication, how did homomers evolve to selectively assemble with copies of themselves (excluding their sister paralogs), and was evolution of new functions constrained until this selectivity was achieved? By combining advanced techniques from protein biochemistry and evolutionary biology, the project will articulate and test at unprecedented resolution hypotheses about the evolutionary forces and biochemical mechanisms that underlie the physical and functional properties of molecular complexes. It will also help to explain why multimers are so widespread and, by revealing how evolution achieved specificity and allosteric regulation, enhance engineering efforts to design complexes with these properties. The project will also provide a methdological and conceptual template for future studies of other molecular complexes.